This invention relates to an in vitro method for the detection of cellular pathology, and more specifically to an assay for monitoring cytoskeleton breakdown products to determine cell necrosis.
This invention was made with government support under AFOSR Contract No. 86-0099 (P.I.: Lynch), NIH Grants Nos. NS-18427 and NIA Grant No. AG00538 The government has certain rights in this invention.
The structural integrity of cells is maintained in part by the cytoskeleton, a mesh-like structure composed primarily of proteins, which lies adjacent to the inner cell surface. The cytoskeleton of many cell types (a partial list includes neurons, lymphocytes, kidney, liver, cardiac and smooth muscle, and blood platelets) contain large amounts of a protein either identical to or closely related to brain spectrin (also known as fodrin). Spectrin binds F-actin, and together they are generally associated with the inner face of the cell membrane, where they form a filamentous meshwork.
Brain and many other tissues have been known for some time to express calcium-stimulated proteolytic activity. Studies of degradation in peripheral nerves have indicated that a calcium activated neutral protease, calpain, is critically involved in the degradation of neurofilament proteins following denervation or injury. Two forms of this protease have been identified in brain and other tissues. The two forms are differentiated by their threshold for activation by calcium: calpain I requires micromolar calcium while calpain II is activated by concentrations of calcium between 0 1 and 0.5 mM. The two forms are differentially distributed in the brain. While calpain II is mainly localized in the cytoplasmic fraction of brain cells, the highest activity of calpain I is found in small processes. While the two forms of calpain differ in these and other ways, the term "Calpain" shall be used herein to refer to calcium activated neutral proteases generally, including both forms of calpain.
A variety of cellular insults (e.g., toxins, anoxia, etc.) and disease states (e.g., Alzheimer's, Parkinson's, HIV-induced neuropathy, muscular dystrophy) cause the degeneration and death of cells. Often, however, it is not possible to determine that injury has occurred until degenerative effects are irreversible. There thus exists a need for reliable methods to detect degenerative events as soon as possible, preferably before the onset of pathological symptoms. Preferably such methods also have high sensitivity, wide ranging applicability and ease of administration.